Inactivation method for snake venom and inactivated snake venom prepared thereby

A snake venom and inactivation technology, applied in the field of snake venom inactivation and toxicity inactivation, can solve the problems of animal immune intolerance, conformational change of snake venom antigen, low neutralizing antibody titer, etc. Reduce antigen dosage and produce rapid results

Inactive Publication Date: 2011-11-16
SHANGHAI SERUM BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This approach makes animals intolerable and has become a bottleneck in our antitoxin production
[0007] In short, formaldehyde inactivated snake venom still preserves some of the toxicity of the snake venom, the conformation of the snake venom antigen changes, and the antigenicity decreases, which leads to the intolerance of animals during immunization and the low titer of neutralizing antibodies obtained

Method used

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  • Inactivation method for snake venom and inactivated snake venom prepared thereby
  • Inactivation method for snake venom and inactivated snake venom prepared thereby
  • Inactivation method for snake venom and inactivated snake venom prepared thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Iodoacetamide method to inactivate viper venom:

[0030] The viper venom was purchased from Guangzhou Institute of Snake Venom, which is a freeze-dried toxin. Accurately weigh 100 mg of lyophilized toxin and dissolve it in 1000 ml of 50mM pH7.8 Tris buffer. After the snake venom is dissolved, centrifuge at 4000 rpm for 30 minutes to remove precipitated particulate matter. The supernatant is slowly poured into 3000 ml of iodoacetamide solution (50mM Tris , 1.33mM EDTA, 2.67M NaCl, 2.67M urea, 266.7mM iodoacetamide (pH 8.0), mix well and place in a 37°C water bath for 3 hours to avoid light. After the reaction, ultrafiltration was performed with a Millipore plate ultrafilter with a molecular weight cut-off value of 3000d. At the same time, the buffer was changed to pH7.0, 50mM Tris, 0.15M sodium chloride buffer, and the final protein concentration was concentrated to 1mg / ml, and stored at low temperature for later use.

Embodiment 2

[0031] Example 2. Inactivation of viper venom by formaldehyde method:

[0032] Take 100 mg of the above freeze-dried snake venom and add 5 ml of phosphate buffer solution to prepare a solution containing 20 mg of snake venom per ml. Add 50 microliters of formalin to the snake venom solution, shake well, and treat at room temperature for 7 days for inactivation ; The obtained inactivated attenuated snake venom is used as an immune antigen.

Embodiment 3

[0033] Example 3. SDS-PAGE comparison of electrophoretic characteristics of inactivated snake venom by two different methods

[0034] Comparison of inactivated snake venom and formaldehyde inactivated SDS-PAGE: After the snake venom was treated with iodoacetamide and dialyzed, it was separated on SDS-PAGE with a voltage of 150V (12.5% ​​separation gel, 4 %Laminated glue)( figure 1 ), the formaldehyde-inactivated snake venom was separated on SDS-PAGE with a voltage of 150V (12.5% ​​separation gel, 4% layering gel) ( figure 2 ), the gel was stained with Coomassie Brilliant Blue R-250 after electrophoretic separation. It can be seen from SDS-PAGE that the formaldehyde-inactivated snake venom forms a high-molecular polymer. figure 1 Compared with the snake venom before and after treatment with iodoacetamide, its molecular weight is significantly increased and dispersed, indicating that the proteins are cross-linked with each other, even in the presence of SDS and reducing agent, hea...

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Abstract

The invention relates to an inactivation method for snake venom. According to the method, snake venom is alkylated by a protein alkylating reagent, and therefore snake venom is inactivated. Preferably, alkylation is carried out under reversible denaturation conditions and finished after lucifugal culturing for 1 to 10 hours at a temperature of 20 to 40 DEG C; the protein alkylating reagent is iodoacetamide, iodacetic acid or iodoacetate; the alkylation is carried out in a buffer solution for snake venom; the concentration of snake venom is 0.01 to 100 mg / ml; and snake venom is venom of viper or pallas pit viper, cobra, long-noded pit viper and coral snakes. The invention also relates to inactivated snake venom prepared by the inactivation method. The method is novel and enables no obvious change of conformation of snake venom and retention of critical antigen epitope; therefore, when inactivated snake venom is immunized, high titer neutralizing antibodies can be generated, the amount of antigen for immunization is reduced, side reactions are avoided, rapid generation of high titer neutralizing antibodies is obtained, and the method is suitable for large scale popularization and application.

Description

Technical field [0001] The invention relates to the technical field of toxicity inactivation, in particular to the technical field of snake venom inactivation, and specifically refers to a method for inactivating snake venom and the inactivated snake venom prepared thereby. Background technique [0002] The research of antivenom has a history of more than 100 years. The first clinical application was the anti-cobra venom serum prepared in India. In the future, there will be many scientists in the world engaged in this research work. At present, more than 30 institutions in more than 20 countries in the world use more than 60 kinds of venomous snake venom to produce antivenoms, and there are nearly a hundred monovalent and multivalent antivenoms in clinical use. However, so far, there are defects of low immune titers, few effective antibodies, and large doses that need to be used, leading to the major problem of large side effects in clinical applications. [0003] As early as 19...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C07K1/107
Inventor 孙九如范志和柏伟杨涛李晓王静马祖严
Owner SHANGHAI SERUM BIOTECH
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